The present invention relates to a process for amplifying nucleic acid sequences by means of the polymerase chain reaction. More specifically, it relates to a process wherein the consecutive cycles of denaturation and renaturation are achieved by a controlled oscillation of the local concentration of divalent metal ions. This allows the reaction to proceed at constant temperature, and depending upon the metal ions used at lower temperatures near physiological values.
In the biotechnology and biomedical industry large number of copies of a particular gene or polynucleic acid may be needed for various purposes such as sequencing and diagnostic applications. Simple and reliable methods to generate such amounts are consequently indispensable for the success of future industrial and scientific developments. Any new technique to amplify genetic material and in particular for diagnostic applications, should minimize human intervention and chemical addition steps. A further prerequisite is that it should be easily amenable to automation.
Currently relatively large amounts of particular gene sequence can be produced by the polymerase chain reaction (EP-B-0200 362, EP-B-0201 184). A method which offered significant benefits over classical procedures such as cloning. In essence PCR is based on the repetitive thermal denaturation of double stranded (dsDNA), a process which is known as thermal cycling. As the reaction temperature is cycled between about 70xc2x0 C. and 94xc2x0 C., the polymerases would denature soon. In Kleppe et al., J.Mol.Biol. 56: 341 (1971), a process is described for synthesizing DNA using primer-initiated, template-directed repair replication, and it is suggested that cycles of replication could be repeated, adding every time a fresh dose of DNA polymerase, which would be an expensive procedure. A nice solution was offered by the introduction of thermostable DNA polymerases which were derived From thermophilic bacteria (e.g. Thermus aquaticus). Such enzymes, however, have a higher error rate than other polymerases, particularly of eukaryotic origin, which operate at lower temperatures.
The need for extreme conditions to allow separation of both strands that make us the DNA helix directly results from the fact that the DNA double helix is a relatively stable structure due to the propensity of the bases to form hydrogen bonds with each other in a very specific way. Apart from base pairing several additional conditions need to be satisfied to guarantee stability at a particular temperature. Ionic strength of the medium has a very important role and at low electrolyte concentration dsDNA is denatured due to the lack of counterions. These counterions may be mono- or divalent metal ions which stabilize the structure by binding to the phosphate moieties and effectively cancel the net negative charges preventing unwinding of the helix due to repulsive forces. However at elevated concentrations some divalent metal ions (in particular: Cu2+, Cd2+, Zn2+ and Mn2+) destabilize the double helical structure. This is because all these ions exhibit an affinity to both phosphate and bases, with their association constants being significantly different, Cu2+ for example has the highest affinity for DNA bases and in particular N-7 of guanine is the prime target for Cu2+ complexation. Hence, when an increasing portion of the phosphata vacancies are filled, the affinity constant for the binding of a particular metal to phosphate decreases as a consequence of the cooperative binding nature. At this point, binding to the DNA bases becomes more important and competition for hydrogen bonding is initiated. The effect is manifested in a lowering of the melting temperature (Tm) of the dsDNA (Eichhorn and Shin, J.Am.Chem.Soc., 90: 7323 (1968) P -Y. Cheng, Biochem. Biophys.Acta., 102: 314 (1962); Schreiber and Daune, Biopolymers, 8: 130 (1969)).
The present invention provides alternative solutions for the above-mentioned problem. Instead of providing thermostabile DNA polymerases to cope with the extremely high temperatures needed to allow the DNA to denature, the present invention provides means for manipulating the conditions such that the DNA can denature at much lower temperatures and consequently no longer draws upon the use of thermostabile DNA polymerases.
It is thus an aim of the present invention to provide an alternative PCR amplification process.
It is also an aim of the present invention to provide an alternative PCR amplification process, that does not draw upon the use of thermostabile DNA polymerases.
It is also an aim of the present invention to provide an alternative type of PCR amplification kits.
It is further an aim of the present invention to provide an alternative type of PCR amplification device.
According to a preferred embodiment, the present invention relates to the use of a controlled oscillation of the concentration of divalent metal ions such as Cu2+, Cd2+, Zn2+ and Mn2+, thereby forming the basis for isothermal denaturation of the double helix.
The present invention also provides means for the automatization of this process. For instance, the new method favours dynamic electrochemical control of the activity of ionic species present. Furthermore only divalent metal ions are considered at this stage. This however, by no means excludes the potential to extend electrolytic control to the activity of mono-valent cations, which are equally important members in terms of contributions to total ionic strength. The present invention departs from methods that allow separation of both strands of a DNA helix. This is not achieved solely by increasing the temperature but also by increasing the local concentration of divalent metal ions that have the tendency to destabilize the DNA helix. Destabilization of the DNA helix is reflected in the lowering of the melting temperature (Tm) of DNA (i.e. the midpoint in the transition of dsDNA to ssDNA). Cu2+ ions stabilize the double helical structure at low concentrations. However, at elevated concentrations Cu2+ ions start to interfere with hydrogen bonding resulting in the transition from double helical into ssDNA.
According to a preferred embodiment the present invention relates to a process for amplifying at least part of a specific double-stranded nucleic acid sequence contained in a sample comprising:
(a) separating the nucleic acid strands in said sample essentially with a means for increasing the local concentration of metal ions, preferably of divalent metal ions;
(b) treating the strands with at least one oligonucleotide primer under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions, and in the presence of an inducing agent for polymerization and the different nucleotides, such that an extension product of the respective primer(s) is synthesized which is complementary to one end of the sequence to be amplified on one of the strands such that an extension product can be synthesized from said primer which, when it is separated from its complement, can serve as a template for synthesis of an extension product of the other primer;
(c) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules essentially with a means for increasing the local concentration of metal ions, preferably divalent metal ions;
(d) treating the single-stranded molecules generated from step (c) with the primers of step (b) under hybridizing conditions essentially with a means for decreasing the local concentration of metal ions, preferably divalent metal ions, and in the presence of an inducing agent for polymerisation and the different nucleotides such that a primer extension product is synthesized using each of the single-strands produced in step (c) as a template; and, if desired;
(e) repeating steps (c) and (d) at least once; whereby the amount of the sequence to be amplified increases exponentially relative to the number of steps in which primer extension products are synthesized.
The term xe2x80x9cessentiallyxe2x80x9d refers to the fact that the nucleic acid strand separation and annealing process according to the present invention is mainly brought about by cycling or fluctuating the metal ion concentrations. This does exclude the influence and concomitant use of other agents and/or parameters for the described process. The term xe2x80x9cseparatingxe2x80x9d has to be interpreted in a broad sense, such that it does not only refer to the actual physical separation of both strands that make up a DNA helix or a template-primer complex, but more to the physical separation of the DNA bases that interact as within a Watson Crick DNA-duplex. In its broadest sense the term xe2x80x9cseparationxe2x80x9d of two strands can be defined operationally as a process which creates a situation, such that annealing of another primer or an oligonucleic acid becomes possible to one of the original strands that made up the original DNA-duplex.
The term primer as used throughout the specification and the claims has to be interpreted in a broad sense. A primer as used in ordinary PCR reactions is usually about 20 basepairs long. However, with respect to the present invention a primer can be much shorter, and many more primers can be used as the usual pair of primers used in an ordinary PCR reaction. Also these primers can be immobilized and/or labelled, such that detection becomes possible. The term primer does not implicate that this oligonucleic acid has to be used in a process in which the primer is extended in a polymerase reaction wherein the complementary strand is used as a template. In this respect the term primer can be more properly defined as an ordinary oligonucleotide.
Besides polymerase chain reactions to amplify DNA, the essential teachings of the present invention, however, also may be applied to other types of reactions involving repetitive denaturation of genetic material. An example of such a reaction is: denaturation of a DNA-duplex according to the methods of the present invention, and concomitant or subsequent annealing according to the methods of the present invention, of a primer or a primer pair or many primers or oligonucleic acids of any type including the use of PNA""s.
The present invention more particularly relates to a nucleic acid amplification process that allows for the controlled cycling of other metal ions than Cu2+. While Mg2+ does not readily bind with the bases of the nucleotides that make up the polynucleic acid polymer, thereby interfering with the hydrogen bonding, other bivalent metal ions like Cu2+, Cd2+, Zn2+ and Mn2+, do exhibit this property, and thus are candidate ions that can be used to substitute for Cu2+. It has to be understood that certain combinations of divalent metal ions are equally well candidates to lower the melting temperature of the DNA helix. By way of example but not limiting for the present invention, a useful combination could be to allow fluctuations of the Cu2+ concentration or of the concentrations of Cd2+, Zn2+ and Mn2+, or to allow fluctuations of the concentrations of combinations of such divalent metal ions (such as Cu2+ and Cd2+, or Cu2+ and Zn2+, or Cu2+ and Mn2+), while the Mg2+ concentration is allowed to fluctuate in reverse sense (see Examples section). This can be helpful in order to destabilize the DNA helix, because Mg2+-ions are known to stabilize the DNA helix. It is also helpful to allow reannealing or renaturation of the DNA helix, or of a primer with a template, because higher Mg2+ concentrations facilitate dissociation of the DNA-ion complex and thus the reversibility of the process.
The concentrations of divalent metal ions used according to the present invention will vary typically from 10xe2x88x922M to 10xe2x88x925M, more preferably from 10xe2x88x923M to 10xe2x88x924M.
According to another embodiment, the present invention also relates to the use or a controlled oscillation of the concentration of Mg2+ ions. Although this process is not based on competition between metal ion and hydrogen bonding and unwinding of dsDNA is solely based on the counter ion effect, it has significant advantages in terms of toxicity to DNA polymerase enzymes that may be used. In this case the temperature for unwinding of the DNA helix, unfortunately needs to be relatively high (about 70xc2x0 C.) This however may be improved by exerting control over the monovalent metal ions present in the reaction medium, or by adding small amounts of divalent cations such as Cu2+, Cd2+, Zn2+ and Mn2+, or by increasing the hydrophobicity of the reaction medium.
The present invention also relates to analogous processes for amplifying single stranded nucleic acids using primers in which the primers are annealed to the nucleic acid by means of metal ion concentration fluctuations as described above and below.
In case Cu2+ is used as a divalent metal ion, the Cu2+ concentration that is initially high to allow strand separation of the DNA helix, can be decreased electrochemically in a controlled way through selective reduction. This can be achieved at relatively high current efficiency as the standard reduction potential of Cu2+ and Mg2+ are separated by about 2.7V. The metal copper that has been deposited on the cathode can be oxidized again towards Cu2+, thereby increasing the Cu2+ concentration to the initial concentration that allows separation of the DNA strands. Successive oxido-reduction cycles of copper, hence provides a means for the cycling process needed for a polymerase chain reaction, namely the successive separation of both strands of the DNA helix and reannealing with appropriate primers, upon which the primers can be extended by using the separated strands from the previous cycle as a template.
The present invention relates to the use of any type of electrode that allows for reduction of Cu2+ or other divalent metal ions as mentioned above. Such process can include the use of a conventional electrolytic cell consisting of a dropping mercury or a rotating disc electrode to establish the kinetics appropriate for the reaction.
The present invention also relates to the use of specific types of electrodes that allow for the selective reduction of Cu2+ or other divalent metal ions as mentioned above, that can be constructed and that can possibly be based on information obtained from the above mentioned electrode. Such electrodes can be based on known technologies such as semi-conductor technology such as IFSET (Ion Selective Field Effect Transistor). A further advantage of electrochemical reduction is that it provides cheap active monitoring system to follow the reaction as electrical signals are generated from the electrode as a reaction product.
A very convenient method to control the concentration of metal ions is by cathodic reduction of the desired ionic species. This does not exclude the possibility that the Cu2+ concentration that was initially high, is restored by adding Cu2+ ions as a substitution for the oxidation of metal Cu0 towards Cu2+.
The present invention also relates to the use of chelating agents (e.g. EDTA) in combination with the above mentioned process. It is anticipated that metal chelators can be employed in order to facilitate diffusion to the cathode and/or dissociation of the copper-DNA complex.
The present invention also relates to a process wherein the above mentioned methods are combined with the successive oxido-reduction of monovalent metal ions, possibly the monovalent metal ions of the above mentioned ions, possibly in combination with chelators known in the art that may be specific for the above mentioned ions.
According to a preferred embodiment, the present invention also relates to the use of several regimens to bring about a cycling of successive denaturation and renaturation of the DNA, wherein several combinations of metal ions, preferably divalent and as mentioned above, possibly in concert with chelators.
By way of example (see Examples section below) but not limiting for the present invention are two regimens, one wherein the Mg2+ concentration is kept constant and one with variable Mg2+ concentration. Constant high Mg2+ concentration limits the binding vacancies for any incoming metal ion to the base pairs as phosphates are saturated, this reduces the concentration of metal needed and consequently speeds up the oxido-reduction cycles. Apart from this, toxic effects due to the metal ions with respect to the polymerase enzymes are kept to a minimum. In those cases where the stabilizing effect of Mg2+ outweighs the destabilization by the respective metal ions, a regimen wherein a cycling magnesium concentration is used might be preferred. In each case, contributions from monovalent metal ions to the stability of duplex DNA should be as low as possible.
According to another preferred embodiment, the present invention also relates to alternative ways to allow for a controlled oscillation of the local metal ion concentration. One possibility is the use of a dialysis system, wherein the polymerase chain reaction components are kept contained from the surrounding medium through a dialysis membrane, and wherein the surrounding medium can be flushed with appropriate solutions, in a cyclic and controlled way, thereby allowing fast exchange of electrolytes, chelators and metal ions between the solutions that are flushing the sealed off system and the solution in which the components for the polymerase chain reaction are contained. Such a procedure allows for the controlled oscillation of the concentration of many compounds. By way of example but not limiting to the present invention, it is anticipated that it might be advantageous to manipulate the concentration of the electrolyte as well.
The present invention also relates to a process for nucleic acid amplification wherein the agent for polymerization is a DNA polymerase which can function at temperatures near physiological values.
The following examples merely serve to illustrate some of the aspects of the present invention. The contents of all mentioned references and patents (particularly all patents relating to PCR) are to be considered as incorporated within the content of the invention.